Summary Despite years of research, it is still unclear which antigen-specific CD4+ T cells initiate T1D. New evidence suggests that hybrid peptides (HP) formed from the fusion of islet ? cell proteins may be critical antigens in T1D as recent studies identified HP-reactive CD4+ T cells from T1D patients and diabetic mice in vitro. In preliminary studies, we identified HP-specific CD4+ T cells using novel tetramer reagents, and showed they can cause T1D in mouse transfer models. Thus, we hypothesize that HPs are critical antigens and that autoreactivity to HPs initiates T1D. The deciding factor in whether HP will prime CD4+ T cells to initiate T1D is the inflammatory context during initial T cell receptor signaling, particularly the timing of type I interferon (IFN-I) exposure. Aim 1 will utilize mouse strains of varying T1D susceptibilities, and evaluate their frequency and activation phenotype of HP-specific cells. We predict that targeting hybrid peptide-specific cells will prevent and possibly reverse T1D, thus confirming their pathogenic role. Completion of this work will also provide insight into the role of hybrid peptide-specific cells in human T1D, as we will evaluate the frequency and phenotype of these cells in T1D patients and at-risk individuals. Aim 2 will test the hypothesis that IFN-I or viral infection(s) concurrent with TCR signaling leads to T1D, while IFN-I exposure preceding TCR signaling promotes Tregs and protection from T1D. Finally, we will test tolerance induction using antigen-coupled cells or novel peptide:MHCII blocking antibodies in normal microbial experience mice to determine if tolerance can be induced in a physiological environment more closely resembling that of humans.